Technical Abstract:
Xylella fastidiosa (Xf) strain RIV11 harbors a 25 kbp plasmid (pXFRIV11) belonging to the incP1 incompatibility group. Replication and stability factors of pXFRIV11 were identified and used to construct plasmids able to replicate in both Xf and Escherichia coli. Sequences required for replication in E. coli and conferring antibiotic resistance were derived from the cloning vector pCR2.1. Replication in Xf required a 1.35 kbp region from pXFRIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). This region also conferred plasmid replication in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Constructs containing the trfA gene and oriV derived from pVEIS01, a similar 31 kbp incP1 plasmid of the earthworm symbiont Verminephropbacter eiseniae, also were competent for replication in Xf. As expected, constructs bearing only trfA or oriV from either incP1 plasmid were unable to replicate in Xf. Although these incP1 replicons could be maintained in Xf under antibiotic selection, removal of selection resulted in loss of the plasmid. A novel toxin/antitoxin (pemI/pemK) addiction system of pXFRIV11 was added, dramatically improving stability of incP1 replicons in Xf in the absence of antibiotic selection. The resulting 6 kbp Xf shuttle vector (pXF20-PEMIK) also contains 10 unique endonuclease recognition sites for insertion of foreign DNA.